Vasoactive Intestinal Peptide Receptor Subtypes Research Articles

Are heterobivalent GRPR- and VPAC1R-bispecific radiopeptides suitable for efficient in vivo tumor imaging of prostate carcinomas?

Receptor-specific peptides labeled with positron emitters play an important role in the clinical imaging of several malignancies by positron emission tomography (PET). Radiolabeled heterobivalent bispecific peptidic ligands (HBPLs) can target more than one receptor type and by this – besides exhibiting other advantages – increase tumor imaging sensitivity. In the present study, we show the initial in vivo evaluation of the most potent heterobivalent gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-bispecific radiotracer and determined its tumor visualization potential via PET/CT imaging. For this purpose, the most potent described HBPL was synthesized together with its partly scrambled heterobivalent monospecific homologs and its monovalent counterparts. The agents were efficiently labeled with 68Ga3+ and evaluated in an initial PET/CT tumor imaging study in a human prostate carcinoma (PCa) xenograft rat tumor model established for this purpose. None of the three 68Ga-HBPLs enabled a clear tumor visualization and a considerably higher involvement in receptor-mediated uptake was found for the GRPR-binding part of the molecule than for the VPAC1R-binding one. Of the monovalent radiotracers, only [68Ga]Ga-NODA-GA-PESIN could efficiently delineate the tumor, confirming the results. Thus, this work sets the direction for future developments in the field of GRPR- and VPAC1R-bispecific radioligands, which should be based on other VPAC1R-specific peptides than PACAP-27.

Expression and function of vasoactive intestinal peptide receptors in human lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is an important neurotransmitter involved in the modulation of gastrointestinal function through the stimulation of VIP receptors. However, the expression of VPAC1R, VPAC2R and PAC1R in the human Lower esophageal sphincter (LES) has not been fully clarified. Therefore, the purpose of this study is to explore the expression of these receptors in the human Lower esophageal sphincter, the responses of the Lower esophageal sphincter to Vasoactive intestinal peptide, and the role of Vasoactive intestinal peptide receptors in the responses. Sling and clasp fiber samples of LES were acquired from patients undergoing subtotal esophagectomy, while circular muscle bundles from the esophagus and gastric fundus were used as control groups. Western blotting and RT-PCR technology were performed to determine the expression of the three VIP receptor subtypes. The isometric tension responses of the muscle sample strips to Ro25-1553 and PG99-465, and the effect of electrical field stimulation (EFS) on the sling and clasp fibers were studied. We found that VPAC2R messenger RNA (mRNA) and protein were expressed in the sling and clasp fibers of human LES. However, no VPAC1R or PAC1R mRNA and protein expressions were found in the LES samples. The sling and clasp fibers of the LES produced significant concentration-dependent relaxation following exposure to Ro25-1553 and EFS could induce them to produce frequency-dependent relaxation. Furthermore, the relaxation responses of the LES were inhibited by PG99-465 and induced by EFS and Ro25-1553. VPAC2R, but not VPAC1R or PAC1R, is expressed by the human LES. The relaxation responses of the LES generated by the VIP receptor agonist Ro25-1553 and EFS could be inhibited by the selective VPAC2 receptor antagonist PG99-465. VPAC2R may be important for the generation of relaxation and functional regulation of the LES.

Adrenalectomy impairs vasoactive intestinal peptide-induced changes in food intake and plasma parameters.

The aim of this study is to evaluate the effects of adrenalectomy (ADX) and glucocorticoid in the changes induced by intracerebroventricular (ICV) administration of vasoactive intestinal peptide (VIP) on food intake and plasma parameters, as well as VIP receptor subtype 2 (VPAC2) mRNA expression in different hypothalamic nuclei of male rats. Male Wistar rats (260-280 g) were subjected to ADX or sham surgery, 7 days before the experiments. Half of ADX animals received corticosterone (ADX + CORT) in the drinking water. Animals with 16 h of fasting received ICV microinjection of VIP or saline (0.9% NaCl). After 15 min: (1) animals were fed, and the amount of food ingested was quantified for 120 min; or (2) animals were euthanized and blood was collected for biochemical measurements. Determination of VPAC2 mRNA levels in LHA, ARC, and PVN was performed from animals with microinjection of saline. VIP treatment promoted the anorexigenic effect, which was not observed in ADX animals. Microinjection of VIP also induced an increase in blood plasma glucose and corticosterone levels, and a reduction in free fatty acid plasma levels, but adrenalectomy abolished these effects. In addition, adrenalectomy reduced mRNA expression of VPAC2 in the lateral hypothalamic area and arcuate nucleus, but not in the paraventricular nucleus. These results suggest that adrenal glands are required for VIP-induced changes in food intake and plasma parameters, and these responses are associated with reduction in the expression of VPAC2 in the hypothalamus after adrenalectomy.

Design, synthesis and in vitro evaluation of heterobivalent peptidic radioligands targeting both GRP- and VPAC1-Receptors concomitantly overexpressed on various malignancies – Is the concept feasible?

Radiolabeled heterobivalent peptidic ligands (HBPLs), being able to address different receptors, are highly interesting tumor imaging agents as they can offer multiple advantages over monovalent peptide receptor ligands. However, few examples of radiolabeled HBPLs have been described so far. One promising approach is the combination of gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-targeting peptides into one single radioligand since gastrinomas, prostate and breast cancer have been shown to concomitantly or complementarily overexpress both receptors. Here we report the design and synthesis of different HBPLs, comprising a GRPR-binding (BBN7-14) and a VPAC1R-targeting (PACAP-27) peptide. The heterodimers were varied with regard to the distance between the peptide binders and the steric rigidity of the systems. We radiolabeled the HBPLs 19–23 as well as their monomeric reference standards 26 and 27 with 68Ga, achieving radiochemical yields and purities of 95–99% and non-optimized molar activities of 25–61 GBq/μmol. We tested the stability of the radioligands and further evaluated them in vitro regarding their uptake in different prostate carcinoma cell lines (PC-3, DU-145 and VCaP cells).We found that the heterobivalent substances [68Ga]19 − [68Ga]23 showed comparable uptakes into the tumor cells to those of the respective monomers [68Ga]26 and [68Ga]27, indicating that both peptides are still able to address their target receptors. Furthermore, the obtained results indicate that in case of overall low receptor densities, heterobivalent peptides surpass peptide monomers in tumor cell uptake. Most importantly, it could be shown by blocking studies that both peptide parts of the HBPL [68Ga]19 contributed to tumor cell uptake in VCaP cells, expressing both receptor types.Thus, we describe here the first examples of HBPLs being able to address the GRPR as well as the VPAC1R and have the potential to − by several mechanisms − improve tumor targeting for several malignancies compared to monospecific peptides.

VPAC1 receptors regulate intestinal secretion and muscle contractility by activating cholinergic neurons in guinea pig jejunum

In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin(+) excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY(+) secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 μM), PG97-269 (VPAC1 antagonist; 1 μM), and hyoscine (10 μM), but not by hexamethonium (200 μM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl(-) secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.

Gene expression of vasoactive intestinal peptide receptors in human lung cancer

Despite significant improvement in the diagnosis and treatment of various human carcinomas, the 5-year survival rate for lung cancer remains below 20%. Vasoactive intestinal peptide (VIP) is an important neuropeptide in the control of lung physiology, and exerts its functions mainly through two receptor subtypes, VPAC1 and VPAC2. Receptors for VPAC1 and VPAC2 are present in human lung cancer cells, but very limited information exists about the mRNA expression of these VIP receptor subtypes in lung cancer specimens. The aim of the present study was to investigate by RT-PCR the mRNA expression of the VPAC1 and VPAC2 receptors in surgical specimens of 43 human lung cancer specimens and 7 normal lung samples. mRNA expression of the VPAC1 receptor was detected in 51% of the tumor specimens, while the incidence of mRNA expression for VPAC2 was 46%. Twenty-one percent of the tumor samples expressed only the VPAC1 receptor and 16% displayed only the VPAC2 receptor, while 13 samples (30%) expressed neither subtype. Thirteen cancer tissue specimens (30%), expressed both of these VIP receptor subtypes. Three normal lung tissue specimens also displayed gene expression for VPAC1 and/or VPAC2 receptors. Our results support the additional investigation of the role of VIP and its receptors in human lung cancer and suggest a further development of VIP analogs for therapeutic and imaging purposes in this malignancy.

Human hepatic progenitor cells express vasoactive intestinal peptide receptor type 2 and receive nerve endings

We recently showed that human hepatic progenitor cells (HPCs) express muscarinic acetylcholine (Ach) receptor subtype 3 and that--following liver transplantation--HPC numbers are significantly reduced. To further elaborate on this, we examined whether HPC also express receptors for vasoactive intestinal peptide (VIP), which, besides Ach, also is an important parasympathetic neurotransmitter. VIP expressing nerves are known to be present in the liver. We performed immunohistochemistry for VIP receptor subtypes 1 and 2 (VIPR1 and 2), on sections of normal and diseased human liver (n=17), and double staining for VIPR2 and known HPC markers. We performed RT-PCR for VIPR1 and 2 on total RNA from purified rat HPC. To document the probability of direct interaction, we also performed double immunostaining for nerve markers and HPC markers on human liver sections. VIPR2 immunostaining was clearly positive in HPC and reactive bile ductules on paraffin-embedded and frozen tissue sections. We could not demonstrate VIPR1 protein expression in the liver, with either of two VIPR1 antibodies tested. The presence of VIPR2 mRNA in HPC was confirmed by RT-PCR. Nerve endings were shown to abut on reactive bile ductules. We show here for the first time that HPC express VIPR2 and receive nerve endings. These features, and the fact that HPC numbers are influenced by the presence or absence of the autonomic innervation of the liver, suggest a direct interaction.

Depolarization and Neurotransmitter Regulation of Vasopressin Gene Expression in the Rat Suprachiasmatic NucleusIn Vitro

Vasopressin (VP) transcription in the rat suprachiasmatic nucleus (SCN) in organotypic culture was studied by in situ hybridization histochemistry using an intron-specific VP heteronuclear RNA probe. The circadian peak of VP gene transcription in the SCN in vitro is completely blocked by a 2 h exposure to tetrodotoxin (TTX) in the culture medium, and this TTX inhibition of VP gene transcription is reversed by exposure of the SCN to either forskolin or potassium depolarization. This suggests that an intrinsic, spontaneously active neuronal mechanism in the SCN is responsible for the cAMP- and depolarization-dependent pathways involved in maintaining peak VP gene transcription. In this paper, we evaluate a variety of neurotransmitter candidates, membrane receptors, and signal-transduction cascades that might constitute the mechanisms responsible for the peak of VP gene transcription. We find that vasoactive intestinal peptide (VIP) and a VPAC2 (VIP receptor subtype 2) receptor-specific agonist, Ro-25-1553, are the most effective ligands tested in evoking a cAMP-mitogen-activated protein kinase signal transduction cascade leading to an increase in VP gene transcription in the SCN. In addition, a second independent pathway involving depolarization activating L-type voltage-gated calcium channels and a Ca-dependent kinase pathway [inhibited by KN62 (1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine)] rescues VP gene transcription in the presence of TTX. In the absence of TTX, these independent pathways appear to act in a cooperative manner to generate the circadian peak of VP gene transcription in the SCN.

Vasoactive intestinal peptide receptor subtypes and signalling pathways involved in relaxation of human stomach

Vasoactive intestinal peptide (VIP) relaxes smooth muscle by interacting with receptors coupled to cAMP- or cGMP-signalling pathways. Their relative contribution to human gastric relaxation is unknown. This study aimed at investigating, in terms of biological activity, receptor expression and related signalling pathways, the action of VIP separately on the human fundus and the antrum. VIP caused greater relaxation of smooth muscle cells (SMC) and strips of the antrum presenting on the former a higher efficacy and potency (ED(50): 0.53 +/- 0.17 nmol L(-1)) than on the fundus (ED(50): 3.4 +/- 1.4 nmol L(-1)). On both fundus and antrum strips, its effect was tetrodotoxin insentitive. Reverse transcriptase-polymerase chain reaction analysis showed the sole expression of VPAC2 and natriuretic peptide clearance receptors, with VPAC2 being more abundant in the antrum. Functional regional differences in receptor-related signalling pathways were found. Activation of the cAMP-pathway by forskolin or its inhibition by adenylate cyclase (2'5'-dideoxyadenosine) or kinase (Rp-cAMPs) inhibitors had more pronounced effects on antrum SMC. Activation of the cGMP-pathway by sodium nitroprusside or its inhibition by guanylate cyclase (LY83583) or kinase (KT5823) inhibitors had more effects on fundus SMC, on which a higher expression of endothelial nitric oxide synthase was found. In conclusion, regional differences in VIP action on human stomach are related to distinct myogenic properties of SMC of the antrum and the fundus.

Coronary vascular effects of vasoactive intestinal peptide in the isolated perfused rat heart.

The potency and mechanism of action of vasoactive intestinal peptide (VIP) for producing coronary vasodilation was investigated in the isolated perfused heart of the rat. VIP reduced coronary vascular resistance in a dose-dependent manner, starting at 1 x 10(-10) M, and maximally reduced coronary vascular resistance by 49% at 1 x 10(-8) M. The potency of VIP for reducing coronary vascular resistance (EC50=3.02 x 10(-10) M) was considerably greater than that of adenosine (EC50=6.17 x 10(-8) M) and sodium nitroprusside (EC50=2.45 x 10(-6) M). The vasodilatory action of VIP was more easily observed after increasing vascular tone by perfusion of the hearts with a modified physiological solution containing reduced concentrations of potassium (3.2 mM) and calcium (1.2 mM). Under these conditions, VIP maximally reduced coronary resistance by 54% at 7 x 10(-9) M. The potency of VIP for reducing coronary resistance in these hearts, however, decreased 16-fold (EC50=4.90 x 10(-9) M) while that of SNP remained unaltered (EC50=3.39 x 10(-6) M), compared with hearts perfused with higher levels of potassium (5.9 mM) and calcium (2.5 mM). The vasodilatory effect of VIP occurred without a significant change in heart rate, myocardial contractility or oxygen consumption. In additional studies, the dose-dependent effect of VIP on cyclic nucleotide release from the heart was determined by infusing VIP into the coronary circulation in a cumulative fashion to produce final concentrations between 1 x 10(-11) and 1 x 10(-9) M. VIP increased cyclic AMP at 1 x 10(-9) M but did not increase cyclic GMP. Studies using RT-PCR and immunohistochemistry clearly demonstrated the presence of two VIP receptor subtypes, VPAC1 and VPAC2, in the arteries and arterioles of the heart. In conclusion, VIP is a potent vasodilator in the coronary circulation of the rat and the role of VIP in the control of coronary vascular resistance depends on the circulating levels of potassium and calcium. This vasodilatory effect involves binding to specific coronary cell surface receptors, VPAC1 and/or VPAC2, and is dependent on cyclic AMP only during maximal vasodilation.

Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Vasoactive intestinal peptide fibers innervate neuroendocrine dopaminergic neurons

Hypothalamic neuroendocrine dopaminergic neurons exhibit a diurnal rhythm. Higher level input to these neurons has not been described. In the present study, we identified fibers known to originate in the suprachiasmatic nucleus (SCN), which were associated with neuroendocrine dopaminergic neurons. Hypothalamic sections were obtained from either ovariectomized (OVX) female rats or OVX female rats implanted with estrogen and progesterone (E+P). Confocal microscopic images were acquired from the periventricular nucleus, as well as the rostral, dorsomedial, ventrolateral, and caudal regions of the arcuate nucleus. Using antibodies directed against vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) the rate-limiting enzyme in dopamine synthesis, fine VIP fibers in close apposition to TH-immunoreactive (IR) soma and proximal dendrites were revealed. Of the antibodies for the two VIP receptor subtypes (VIP1R and VIP2R), only VIP2R was found on TH-IR neurons. E+P significantly increased the incidence and density of neuroendocrine dopaminergic neurons expressing VIP2R, when compared to OVX animals. E+P did not affect the percent of neuroendocrine dopaminergic neurons associated with VIP fibers. No VIP fibers or VIP2R were found on dopaminergic neurons in the zona incerta. Brain sections triple labeled for Synapsin (a protein localized in synaptic vesicles) VIP, and TH demonstrated that Synapsin was colocalized with VIP fibers that were associated with TH-IR neurons in the arcuate nucleus. Double-label immuno-electron microscopy of hypothalamic sections labeled with antibodies for VIP and TH revealed VIP boutons associated with TH-IR soma and proximal dendrites. These results suggest VIPergic neurons may directly regulate neuroendocrine dopaminergic neuron activity, and ovarian steroids may play a modulatory role.

Mutational analysis of the human vasoactive intestinal peptide receptor subtype VPAC(2): role of basic residues in the second transmembrane helix.

1. We investigated the role of two conserved basic residues in the second transmembrane helix arginine 172 (R172) and lysine 179 (K179) of the VPAC(2) receptor. 2. Vasoactive intestinal polypeptide (VIP) activated VPAC(2) receptors with an EC(50) value of 7 nM, as compared to 150, 190 and 4000 nM at R172L, R172Q and K179Q-VPAC(2) receptors, respectively. It was inactive at K179I mutated VPAC(2) receptors. These results suggested that both basic residues were probably implicated in receptor recognition and activation. 3. The VPAC(2)-selective VIP analogue, [hexanoyl-His(1)]-VIP (C(6)-VIP), had a higher affinity and efficacy as compared to VIP at the mutated receptors. 4. VIP, Asn(3)-VIP and Gln(3)-VIP activated adenylate cyclase through R172Q receptors with EC(50) values of 190, 2 and 2 nM, respectively, and through R172L receptors with EC(50) values of 150, 12 and 8 nM, respectively. Asn(3)-VIP and Gln(3)-VIP behaved as partial agonists at the wild type receptor, with E(max) values (in per cent of VIP) of 75 and 52%, respectively. In contrast, they were more efficient than VIP (E(max) values of 150 and 150% at the R172Q VPAC(2) receptors, and of 400 and 360% at the R172L receptors, respectively). These results suggested that the receptor's R172 and the ligand's aspartate 3 are brought in close proximity in the active ligand-receptor complex. 5. The K179I and K179Q mutated receptors had a lower affinity than the wild-type receptors for all the agonists tested in this work: we were unable to identify the VIP amino acid(s) that interact with K179.

Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting.

Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with a wide range of biological activities. Receptors for VIP (VIP-R) are overexpressed in breast cancer, where they may have diagnostic and therapeutic implications. Although N-methyl nitrosourea (MNU)-induced breast cancer in rats is used extensively as a model to study mammary carcinogenesis, there is no information about the expression of VIP-R in this model. Therefore, the purpose of this study was to investigate the presence of VIP-R in MNU-induced breast cancer in rats so that this model can be used to perform studies involving VIP-R. Breast cancer was induced in 36-day-old virgin female Sprague-Dawley rats, by a single intravenous injection of MNU (50 mg/kg body weight). The breast tumors were detected 100-150 days after injection. The normal and cancerous rat breast tissue were excised and 20 micro sections were incubated with 40 nM fluorescein-labeled VIP (Fluo-VIP(TM)), in the presence and absence of 1000-fold excess unlabeled VIP, pituitary adenylate cyclase activating polypeptide (PACAP) or secretin. The sections were visualized under a fluorescence microscope and photographed. Fluo-VIP(TM) stained rat breast cancer tissue homogeneously and to a much greater extent than normal rat breast tissue (p < 0.05). This staining was specific as indicated by displacement of Fluo-VIP(TM) by excess unlabeled VIP and PACAP. Displacement of Fluo-VIP(TM) by secretin indicated the probable presence of VIP receptors of type VPAC1 (VIP receptor subtype 1) in the rat breast. These data suggest that, as in human breast cancer, VIP-R, predominantly of type VPAC1, are overexpressed in MNU-induced rat breast cancer tissue as compared to the normal rat breast tissue. Thus, MNU-induced rat breast cancer model can be used as a tool to study the functional role of VIP-R in human mammary carcinogenesis and VIP-R mediated active breast cancer targeting. This could have implications in the diagnosis, prognosis and therapy of human breast cancer.

Expression and fine mapping of murine vasoactive intestinal peptide receptor 1.

Vasoactive intestinal peptide (VIP) plays multiple roles in the nervous, endocrine, and immune systems as a neurotransmitter, a hormone, and a cytokine. VIP is widely distributed in neurons of the central and peripheral nervous systems (CNS/PNS), and recently has been found to be an important neuroprotective agent. VIP actions are mediated through specific G protein-coupled receptors. We have cloned the cDNA of VIP receptor subtype 1 (VIPR1 or VPAC1) and have demonstrated the quantitative expression profile in mice. Fluorometric real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that VPAC1 is expressed in all tissues examined. Expression was highest in the small intestine and colon followed by the liver and brain. The high level of VPAC1 expression in forebrain and cerebellum suggests that VPAC1 may mediate the neuroprotective effect of VIP. We have refined the chromosomal localization of the mouse, rat, and human VPAC1 genes. This fine mapping of the VPAC1 gene extends the respective regions of synteny between the distal region of mouse chromosome 9, rat chromosome 8q32, and human chromosome 3p21.33-p21.31. Thus, VPAC, constitutes a functional-positional candidate for the tumor-suppressor function mapped to human 3p22-p21 where loss-of-heterozygosity is observed in small-cell lung carcinoma (SCLC) cell lines and primary tumors. Availability of the cDNA sequences for mouse VPAC1 will facilitate the generation of VPAC1 null mutant animals. Such studies will ultimately enhance our understanding of the role of VIP in the nervous system.

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear ( r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC 1 (47 kDa primarily) and VPAC 2 (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.

Microisolated Mouse Osteoclasts Express VIP-1 and PACAP Receptors

Skeletal tissue contains a network of nerve fibers expressing several neuropeptides, including vasoactive intestinal peptide (VIP) and the related peptide pituitary adenylate cyclase activating peptide (PACAP). These peptides have been demonstrated to regulate osteoclast formation and osteoclast activity. Using atomic force microscopy and by analysing changes of the intracellular calcium concentrations, we have recently demonstrated that multinucleated rat osteoclasts have cell membrane binding sites recognising VIP and PACAP. In the present study, we have further studied the expression of VIP receptor subtypes in mouse bone marrow cultures and isolated osteoclasts. A micromanipulation technique was used to isolate pure populations of osteoclasts formed in PTH-stimulated mouse bone marrow cultures. By reverse transcriptase polymerase chain reaction (RT-PCR), we studied the expression of mRNA for VIP-1, VIP-2, and PACAP receptors. The purity of the microisolated osteoclasts was determined by studying the expression of specific mRNA associated with the phenotypic trait of osteoclasts or osteoblasts/stromal cells. In this study, we show that mouse osteoclasts express VIP-1 and PACAP, but not VIP-2, receptor mRNA.

T cell vasoactive intestinal peptide receptor subtype expression differs between granulomas and spleen of schistosome-infected mice.

Abstract Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma mansoni. Vasoactive intestinal peptide (VIP) is a neurokine that can modulate aspects of the immune response by acting through receptors within the granuloma. Cloned are two novel VIP receptor (VIPR) mRNAs (VIPR1 and VIPR2) that also bind a second neurokine called pituitary adenylated cyclase-activating polypeptide (PACAP). The objective of this study was to determine if granulomas express either VIPR1 or VIPR2. Using a radioligand-binding assay, it was established that PACAP is as effective as VIP at displacing radiolabeled VIP from splenocytes and granuloma cells, and that most if not all VIPRs in the spleen and granulomas bind PACAP. PCR amplification of reverse transcribed RNA determined that granulomas express both VIPR1 and VIPR2 mRNAs. Gel electrophoresis and nucleotide sequencing confirmed the authenticity of the PCR products. Also, both receptor subtypes were amplified from several granuloma CD4+ T cell lines; yet reverse transcribed RNA from T cell-depleted, dispersed granuloma cells had only VIPR1 RNA. It is notable that reverse transcriptase-PCR detected only VIPR1 in the thymus and spleen, which are organs rich in T lymphocytes. Thus, the granulomas and spleens from mice with schistosomiasis contain cells that display authentic VIP/PACAP receptors. Moreover, these data suggest that T cells in different compartments vary in VIPR subtype expression. VIPR1 and VIPR2 may have different physiologic roles in inflammation.

Regional distribution of guanine nucleotide-sensitive and guanine nucleotide-insensitive vasoactive intestinal peptide receptors in rat brain

Based on the ability of guanine nucleotides to inhibit the binding of vasoactive intestinal peptide to its receptors, a guanosine 5′-triphosphate analog, guanylyl-imidodiphosphate, was used to differentiate two subtypes (or different functional states of a single subtype) of vasoactive intestinal peptide receptor in brain with in vitro autoradiography. In most brain regions, guanylyl-imidodiphosphate reduced vasoactive intestinal peptide binding between 40 and 60%. However, in the supraoptic nucleus, locus coeruleus, interpeduncular nucleus, facial nucleus, olfactory tubercle and periventricular hypothalamic nucleus, 80% or more of vasoactive intestinal peptide binding was inhibited. In other brain regions, including the medial geniculate, olfactory bulbs, and ventral thalamic nuclei, guanylyl-imidodiphosphate had little effect on vasoactive intestinal peptide binding. In liver, lung and intestine it also partly inhibited vasoactive intestinal peptide binding. Electrophoretic analysis of vasoactive intestinal peptide, covalently cross-linked to its receptors in brain membranes, revealed a pair of bands between 44,000 and 52,000 mol. wt, a component at 64,000 mol. wt and another at 92,000 mol. wt. All were displaceable with vasoactive intestinal peptide but guanylyl-imidodiphosphate displaced only the 64,000 mol. wt band suggesting that the GTP-sensitive vasoactive intestinal peptide receptor seen in brain sections has a molecular weight of about 61,000. The differential sensitivity to guanylyl-imidodiphosphate suggests the existence of at least two vasoactive intestinal peptide receptor subtypes in brain, with distinct regional distribution, and may reflect differential coupling to second messenger systems.

Regulation of secretion by vasoactive intestinal peptide in isolated perfused rat submandibular glands

The isolated, perfused gland was used to examine the regulation of saliva volume and protein content by vasoactive intestinal peptide (VIP). In the absence of other secretagogues, VIP produced a modest, sustained saliva flow with a biphasic dose-response curve in which saliva volume was greatest at 1 nM VIP (28.5 ± 3.8 μl in the first 5 min, n = 4) but reduced at lower and higher concentrations. The protein concentration in saliva released in response to VIP (0.86 ± 0.13 μg/μl) was substantially higher than with 30 nM acetylcholine (0.06 ± 0.02 μg/μl) or 1 nM substance P (0.30 ± 0.05 μg/ μl). During the first 5 min of stimulation, VIP and substance P were synergistic in terms of volume and protein content whereas inclusion of VIP did not increase acetylcholine-stimulated flow in the first 5 min but produced a higher sustained flow over the next hour. After stimulation with acetylcholine, subsequent addition of VIP transiently enhanced saliva volume and protein content in a monophasic, dose-dependent manner with effects at 1 pM VIP and higher. The responses were different for VIP compared with other cAMP-mobilizing agents and the involvement of multiple VIP receptor subtypes was suggested from experiments in which a VIP antagonist blocked the VIP enhancement of saliva volume but not the increase in protein.